This Project examines cardiac injury induced by members of the class of small molecule tyrosine kinase inhibitors (TKIs). These drugs target tyrosine kinases, mutations or amplications of which are causal or strongly contributory in various solid tumors and leukemias. They have revolutionized the treatment of a number of these malignancies, converting uniformly fatal malignancies to chronically manageable, if not curable, diseases. Many of these mutated kinases activate pathways that promote proliferation or prevent apoptosis, and the drugs that inhibit them, therefore, lead to cell cycle arrest or cell death of the cancerous cells. However, all of these mutated tyrosine kinases have wild-type counterparts, many of which are expressed in cardiomyocytes and/or in cardiac-resident stem/progenitor cells (CRSCs). This raises concerns that inhibition of wild-type kinases in the heart might have adverse consequences on cardiomyocytes, possibly leading to cardiac dysfunction and even heart failure. Indeed, our clinical collaborators have recently identified cardiotoxicity of two of these agents that are FDA-approved: imatinib (Gleevec) and sunitinib (Sutent). These findings led us to studies in cultured cardiomyocytes and mouse models that have identified molecular mechanisms by which imatinib induces cardiotoxicity. In Specific Aim 1 we will determine the mechanisms by which sunitinib and another agent, sorafenib (Nexavar), two "multi-targeted" TKIs that not only block angiogenesis but also induce apoptosis of cancer cells, induce cardiac dysfunction. These studies are essential because they will 1) identify the specific target of the TKI, inhibition of which induces cardiomyocyte dysfunction and/or death, and 2) identify the signaling pathway(s) activated in response to the TKIs that mediate dysfunction and death. By identifying the specific target and the pathways mediating toxicity, we should be able to: a) predict cardiotoxicity of future agents that also inhibit that target; b) impact future drug design since in some instances the target, inhibition of which mediates toxicity, is not essential for tumor cell killing and the TKI can be modified to avoid these "bystander targets; c) identify novel strategies for cardioprotection in cases such as with imatinib in which the signaling pathway mediating toxicity is not central to tumor cell killing (e.g. JNKs mediate imatinib toxicity, and thus strategies to inhibit JNKs could reduce imatinib cardiotoxicity but leave tumor cell killing intact); d) alert clinicians and regulatory agencies to potential problems with agents still in development (e.g. lestaurtinib, a JAK2 inhibitor), encouraging them to prospectively examine LV function in late phase clinical trials or early post-FDA approval. The second half of this project focuses on mechanisms of repair following withdrawal of TKI therapy. We have found in the case of imatinib and sunitinib, that recovery of LV function in patients is quite dramatic after stopping the TKIs. Furthermore, in studies in mice, we have found clear-cut evidence of myocyte dropout (decline in LV mass), yet there was little evidence of excessive apoptosis, necrosis, or autophagy. It is our hypothesis that at least part of the toxicity and its reversibility are due to adverse effects of TKIs on key functions of CRSCs. Specifically, our preliminary studies will show that imatinib, which in addition to Abl also inhibits c-Kit (the receptor for stem cell factor), which is expressed on many CRSCs, markedly reduces the ability of c-Kit+ CRSCs in culture to differentiate into cardiomyocytes. Furthermore, imatinib also blocks proliferation and induces apoptosis of c-Kit(-) CRSCs (cardiac side population or SP cells). Thus in two different lineages of CRSCs, imatinib leads to dysregulation of proliferation, differentiation, and apoptosis. We will employ lentivirus-mediated gene transfer of imatinib-resistant mutants of kinases targeted by imatinib (Abl, PDGFRs, and c-Kit) to attempt to "rescue" these CRSCs from the adverse effects of imatinib, thereby identifying the critical target(s) regulating these processes. These studies should not only identify novel mechanisms of TKI cardiotoxicity, but also define factors and pathways regulating these critical CRSC functions, thereby advancing our understanding of basic stem cell biology. Finally, we will correlate these findings in vitro with studies in vivo, examining CRSC proliferation and new myocyte formation. Taken together, we believe these studies will shed light on a burgeoning problem in cancer treatment, with the hope being that with early identification and treatment of patients with TKI-induced cardiotoxicity, patients may be able to be maintained on these life-saving therapies.